Assay for human DNA for gender determination
View Patent ↗A method for determining gender from a human DNA sample. The loci of Alu element insertion is selected, amplified and evaluated in terms of size of the fragment. The gender assay utilizes AluSTXa for the X chromosome, AluSTYa for the Y chromosome, or both AluSTXa and AluSTYa, to reduce the possibility of error to a negligible quantity. The inserted chromosome yields a large fragment when the homologous region is amplified. The males are distinguished as having two DNA amplicons present, while females have only a single amplicon. The kit adapted for carrying out the method includes a pair of primers to amplify the locus and optionally polymerase chain reaction regents.
1. A method for determining gender from a human DNA sample, said method comprising:
providing a human DNA sample, said DNA sample containing X chromosomal material and potentially containing Y chromosomal material;
amplifying at least one locus containing one of AluSTYa locus and AluSTXa locus of the DNA sample in an amplification reaction, wherein the product of the reaction is a mixture of amplified alleles from the amplified locus present in the sample; and
determining the gender of the DNA sample by evaluating the amplified alleles in terms of size and number.
2. The method of claim 1 , wherein said amplification reaction is a polymerase chain reaction.
3. The method of claim 2 , wherein said at least one locus is AluSTYa locus.
4. The method of claim 3 , wherein said amplification step comprises the step of using primer pairs containing the following sequences:
Forward
5′-CATGTATTTGATGGGGATAGAGG-3′
(SEQ ID NO:1)
and
Reverse
5′-CCTTTTCATCCAACTACCACTGA-3′.
(SEQ ID NO:2)
5. The method of claim 2 , wherein said at least one locus is AluSTXa locus.
6. The method of claim 5 , wherein said amplification step comprises the step of using primer pairs containing the following sequences:
Forward
5′-TGAAGAAATTCAGTTCATAGCTTGT-3′
(SEQ ID NO:3)
and
Reverse
5′-CAGGAGATCCTGAGATTATGTGG-3′.
(SEQ ID NO:4)
7. The method of claim 2 , wherein said amplification step comprises an amplification of AluSTXa locus and an amplification of AluSTYa.
8. The method of claim 7 , wherein said amplification step comprises the step of using primer pairs containing the following sequences: for said amplification of the AluSTXa locus,
5′-TGAAGAAATTCAGTTCATAGCTTGT-3′
(SEQ ID NO:3)
and
5′-CAGGAGATCCTGAGATTATGTGG-3′;
(SEQ ID NO:4) and
for said amplification of the AluSTYa,
5′-CATGTATTTGATGGGGATAGAGG-3′
(SEQ ID NO:1)
and
5′-CCTTTTCATCCAACTACCACTGA-3′.
(SEQ ID NO:2)
9. The method of claim 2 , wherein at least one primer of each pair has a fluorescent label covalently attached thereto.
10. The method of claim 1 , wherein the sizes of the amplified alleles are evaluated by fragment resolution on an agarose gel.
11. The method of claim 10 , wherein the sizes of the amplified alleles are evaluated by comparison with a size standard such that:
for a Y insertion AluSTYa an Alu filled site for Y chromosome has size approximately 528 base pairs and an empty site for X chromosome has size approximately 199 base pairs; or
for an X insertion AluSTXa an Alu filled site for X chromosome has size approximately 878 base pairs and an empty site for Y chromosome has size approximately 556 base pairs.
12. The method of claim 1 , wherein male gender is characterized by presence of two DNA fragments and female gender is characterized by presence of one DNA fragment, for amplified loci.
13. A method for quantitating male DNA in a sample, said method comprising the steps of:
amplifying at least one locus of AluSTYa and AluSTXa in the sample by a polymerase chain reaction, wherein said locus is in a non-combining X-Y homologous region and contains a monomorphic Alu insertion;
detecting the product of the amplified locus; and
comparing the detected result with a result of standard male DNA to quantitate DNA in a sample.
14. The method of claim 13 , wherein said amplification step comprises the step of using primer pairs containing at least one of the following pair of sequences:
5′-TGAAGAAATTCAGTTCATAGCTTGT-3′
(SEQ ID NO:3) and
5′-CAGGAGATCCTGAGATTATGTGG-3′;
(SEQ ID NO:4) and
5′-CATGTATTTGATGGGGATAGAGG-3′
(SEQ ID NO:1) and
5′-CCTTTTCATCCAACTACCACTGA-3′.
(SEQ ID NO:2)